Establishment Of Reference Sequences Of Hepatitis B Virus Genotype C Subgenotypes

Background Hepatitis B virus genotype C(HBV/C) has the largest number of subgenotypes(C1-C16) that vary with geography and isolates, nucleotide variation is found intra and inter genotype/subgenotype. Suitable reference strains for different subgenotypes could greatly facilitate research on multiple aspects such as quasispecies, mutation, clinical features, therapy efficacy, but unfortunately they are scarce. Most of the reported reference sequences were either determined from the first isolated strain or established from a limited number of isolates, thus they are not fully fulfil the requirements of HBV research. In this study, we aimed to analyze large number of full-length sequences and establish reference sequences of HBV C subgenotypes to promote researches on the epidemiological and virological features of HBV/C.Methods 974 HBV/C full-length sequences retrieved from the GenBank database were subgenotyped by phylogenetic analysis and then grouped in terms of locations and subgenotypes. Reference sequences of each subgenotype from different locations were established with the most frequent nucleotide present at each position of the isolates that belonged to the same subgenotype. Homology analysis was performed between newly established reference sequence and isolates belonging to the corresponding subgenotype. The frequency of base substitution was analyzed at each position especially in hotspot positions. Homology analyses at the nucleotide and amino acid levels at nucleotide and amino acid levels were performed between newly and previous reference sequences and divergence time of subgenotypes was calculated.Results 1. The reference sequences of subgenotypes C1, C2, C5, and C6 have been constructed and deposited in Gen Bank(KM999990-KM999993). The homology between the reference sequences and almost all the isolates belonging to the corresponding subgenotype was higher than 96%. Similarly, bootstrap values in phylogenetic trees supported clustering of reference strains with isolates belonging to the same subgenotypes. Although the previous C reference sequence was found to belong to HBV/C2, newly established C2 reference sequence, which was phylogenetically more similar to C2 strains, was more representative of the subgenotype. Moreover, both homology and phylogeny analyses showed that reference sequences had significant heterogeneity with isolates from other genotypes and subgenotypes.2. Homology analyses at the nucleotide and amino acid levels at nucleotide and amino acid levels revealed that C1、C2、C5 and C6 shared greater similarity with C than Caus, while C5 has the lowest homology with Caus(92.29%). The homology of S genes among the HBV/C reference strains was slightly higher than among the full-length genomes, while P gene showed the least homology in all four open reading frames(ORFs).3. Homology analysis of complete genomes and S genes between reference strains showed that C1、C5 and C6 were most closely related to HBV/C2. HBV/C2 showed greatest similarity to C1, while C1, C2, and C6 had the lowest homology with C5. Similarly, phylogenetic analysis found HBV/ C5 has the oldest ancestral age, followed by C6, which is much more ancient than C1 and C2.4. Sequence analysis further revealed that mutations were commonly found in the basal core promoter(BCP) region and pre-core(Pre-C), and the substitution rate in BCP was significantly higher than in Pre-C. The rate of A1762T/G1764 A in BCP was extremely high in HBV/C2, relatively high in HBV/C1, but lower in HBV/C5 and HBV/C6. Mutations in nucleotide positions 1858 and 1896 were only found in C1 and C2, while no mutations was found in nucleotide positions 1814 in each subgenotype.Conclusions This study successfully established references for subgenotypes C1, C2, C5, and C6. Homology and phylogeny analyses together proved that our newly established reference strains share greatest similarity with isolates belonging to the same sugbenotype and significant heterogeneity with isolates from other genotypes and subgenotypes, suggesting that established reference sequences of C1, C2, C5, and C6 are highly reliable and representative. The newly established reference sequences of HBV/C may contribute to researches on genetic variations, genetics, pathogenicity, vaccine development and therapeutic intervention.